Compound hydrogel dressing based on recombinant mussel mucin and preparation method thereof

Abstract

The invention relates to the technical field of medical dressings, and discloses a compound hydrogel dressing based on recombinant mussel mucin and a preparation method of the compound hydrogel dressing. The compound hydrogel based on the recombinant mussel mucin is prepared from the following raw material components in parts by weight: 0.01 to 0.3 part of the recombinant mussel mucin, 0.1 to 2.0 parts of a thickening agent, 10 to 21 parts of a polysaccharide composition, 1 to 8 parts of a humectant, 0.01 to 0.5 part of a pH (Potential of Hydrogen) regulator, 0.05 to 0.2 part of a preservative, 0.05 to 0.2 part of a metal ion chelating agent and 68 to 88 parts of purified water, the polysaccharide composition is prepared from glucan, chitosan and hyaluronic acid. The polysaccharide is compounded into the hydrogel of the recombinant mussel mucoprotein to play a role synergistically, and the polysaccharide and the recombinant mussel mucoprotein are closely combined and cross-linked, so that the adhesion of the compound hydrogel dressing is effectively improved, wound healing is promoted, the anti-inflammatory effect is improved, and meanwhile, a good protective barrier environment is formed for wound repair and healing.

Description

Technical Field

[0001] The invention relates to the technical field of medical dressings, in particular to a compound hydrogel dressing based on recombinant mussel mucin and a preparation method thereof. Background Art

[0002] Due to its molecular characteristics, mussel mucin is rich in dopa groups and carries a large amount of positive charge. It has biological activities such as promoting wound healing, anti-oxidation, and anti-inflammation, and has strong biocompatibility and low cytotoxicity. The gene sequence of mussel mucin is introduced into suitable host cells (such as yeast, bacteria, etc.), and recombinant mussel mucin is prepared through fermentation and purification processes. This protein is highly similar to natural mussel mucin in molecular structure and biological activity, but its production process is more controllable and the cost is relatively controllable. However, due to the complexity of the extraction and purification process, the production cost of mussel mucin is still high.

[0003] Therefore, a substance or composition can be found to be compounded with recombinant mussel mucin, which has good biocompatibility, improves the adhesion of the compound and its efficacy in cell repair and anti-inflammatory, reduces the amount of recombinant mussel mucin added, and reduces costs. By effectively integrating this compounded recombinant mussel mucin into hydrogel dressings, new medical dressings with excellent adhesion, biocompatibility and functionality can be developed. These dressings are expected to play an important role in the fields of trauma care, chronic wound treatment, etc.

[0004] However, the existing compound hydrogel wound dressings have insufficient adhesion, which is not conducive to covering skin wounds. In addition, the compound hydrogels have low anti-inflammatory effects on local damaged skin cells, which is not conducive to wound healing and repair. Therefore, it is necessary to find a compound hydrogel dressing that can improve adhesion and the efficacy of the complex, so as to facilitate the integration of recombinant mussel mucin and hydrogel dressings, and promote the wider application of mussel mucin hydrogel dressing products in skin wounds and cell repair dressings.

[0005] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a compound hydrogel dressing based on recombinant mussel mucin and a preparation method thereof, aiming to solve the problems that the existing compound hydrogel dressings have insufficient adhesion, low anti-inflammatory effect on local damaged skin cells and are not conducive to wound healing and repair.

[0007] The technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides a composite hydrogel dressing based on recombinant mussel mucin, which comprises the following raw material components in parts by weight: 0.01-0.3 parts of recombinant mussel mucin, 0.1-2.0 parts of thickener, 10-21 parts of polysaccharide composition, 1-8 parts of moisturizer, 0.01-0.5 parts of pH regulator, 0.05-0.2 parts of preservative, 0.05-0.2 parts of metal ion chelator and 68-88 parts of purified water;

[0009] The polysaccharide composition comprises dextran, chitosan and hyaluronic acid.

[0010] Optionally, the mass ratio of dextran, chitosan and hyaluronic acid is: (5.0-10.0):(5.0-10.0):(0.1-0.5).

[0011] Optionally, the molecular weight of the dextran is 400-600 kDa, the molecular weight of the chitosan is 40-60 kDa, and the molecular weight of the hyaluronic acid is 8-12 kDa.

[0012] Optionally, the thickener is one or more of carbomer, sodium polyacrylate, polyvinyl alcohol, methyl cellulose, hydroxyethyl cellulose, xanthan gum and gelatin.

[0013] Optionally, the humectant is one or more of glycerol, propylene glycol, butylene glycol, pentylene glycol, sorbitol and polyethylene glycol.

[0014] Optionally, the pH adjuster is one of triethanolamine, aminomethyl propanol, sodium hydroxide and potassium hydroxide.

[0015] Optionally, the preservative is one or more of sodium benzoate, benzyl alcohol, methylparaben, sodium methylparaben, sodium propylparaben, phenoxyethanol and potassium sorbate.

[0016] Optionally, the metal ion chelator is one or more of low molecular weight dextran, ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate and sodium calcium ethylenediaminetetraacetate.

[0017] The second aspect of the present invention provides a method for preparing the composite hydrogel dressing based on recombinant mussel mucin, comprising the steps of:

[0018] S1, adding the recombinant mussel mucin freeze-dried powder into water to obtain a recombinant mussel mucin solution;

[0019] S2. Add water, thickener and polysaccharide composition into a vacuum homogenizing stirring tank, heat to 60-85° C., and stir at 200-350 rpm for 20-70 minutes to obtain a first mixed solution;

[0020] S3, adding a moisturizer and a metal ion chelating agent to the first solution, stirring and mixing at a rate of 200-400 rpm, and homogenizing at a stirring rate of 2000-5000 rpm for 5-15 minutes to obtain a second mixed solution;

[0021] S4, cooling the second mixed solution to 30-40° C., adding the recombinant mussel mucin solution, and stirring and mixing at a speed of 200-400 rpm to obtain a third mixed solution;

[0022] S5. Add a preservative to the third mixed solution, stir and mix, then add a pH adjuster and water, stir and mix at 200-400 rpm to obtain the composite hydrogel dressing based on recombinant mussel mucin.

[0023] Optionally, the pH value of the composite hydrogel dressing based on recombinant mussel mucin is between 5.0 and 6.5.

[0024] Beneficial effects:

[0025] The present invention prepares the composite hydrogel dressing based on recombinant mussel mucin by scientifically proportioning the raw material components in the formula. The lysine and dopa groups in the recombinant mussel mucin can attract negatively charged cell or tissue surfaces through electrostatic interactions, thereby promoting adhesion, and exerting its biological activities such as promoting wound healing, anti-oxidation, and anti-inflammation; the scientifically proportioned polysaccharide composition has good biocompatibility, film-forming properties, and viscosity, and has certain antibacterial and anti-inflammatory effects. The present invention compounds polysaccharides into the hydrogel of recombinant mussel mucin, and the polysaccharides work synergistically. Cross-linking occurs between the recombinant mussel mucin and the polysaccharide, and the two are closely combined, which effectively improves the adhesion of the composite hydrogel dressing, promotes wound healing, and improves the anti-inflammatory effect, while forming a good protective barrier environment for the repair and healing of the wound surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of a skin wound deep to the superficial dermis created by surgery at the center of the back of a SD rat in an embodiment of the present invention.

[0027] Figure 2 The figures show the wound healing effects on the 0th, 7th and 14th days after the wound was healed after the composite hydrogel dressing based on recombinant mussel mucin prepared in Example 1 of the present invention was applied to the wound.

[0028] Figure 3 The figures are wound healing effects on the 0th, 7th and 14th days after the wound was healed after the composite hydrogel dressing based on recombinant mussel mucin prepared in Comparative Example 1 of the present invention was applied to the wound.

[0029] Figure 4The figures are wound healing effects on the 0th, 7th and 14th days after the wound was healed after the composite hydrogel dressing based on recombinant mussel mucin prepared in Comparative Example 2 of the present invention was applied to the wound.

[0030] Figure 5 The figures are wound healing effects on the 0th, 7th and 14th days after the wound was healed after the composite hydrogel dressing based on recombinant mussel mucin prepared in Comparative Example 3 of the present invention was applied to the wound. DETAILED DESCRIPTION

[0031] The present invention provides a composite hydrogel dressing based on recombinant mussel mucin and a preparation method thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] The embodiment of the present invention provides a composite hydrogel dressing based on recombinant mussel mucin, which comprises the following raw material components in parts by weight: 0.01-0.3 parts of recombinant mussel mucin, 0.1-2.0 parts of thickener, 10-21 parts of polysaccharide composition, 1-8 parts of moisturizer, 0.01-0.5 parts of pH regulator, 0.05-0.2 parts of preservative, 0.05-0.2 parts of metal ion chelator and 68-88 parts of purified water; the polysaccharide composition comprises dextran, chitosan and hyaluronic acid.

[0034] The recombinant mussel mucin is prepared by introducing the gene sequence of mussel mucin into a suitable host cell (such as yeast, bacteria, etc.) and fermenting and purifying the mussel mucin. Its adhesion mechanism mainly depends on the lysine and dopa groups in its molecular structure. Lysine has a high isoelectric point and carries a positive charge at the physiological pH value of the human body. It can attract negatively charged cells or tissue surfaces through electrostatic interactions, thereby promoting adhesion and exerting biological activities such as promoting wound healing, anti-oxidation, and anti-inflammatory.

[0035] Polysaccharide molecules usually exist in the form of irregular coils in the solution. The hydrophilic groups such as hydroxyl and carboxyl groups on these molecular chains can form hydrogen bonds with water molecules or other substances, so that the polysaccharides have a certain degree of adhesion. And some polysaccharides such as chitosan, dextran, hyaluronic acid, sodium alginate, fucose, trehalose, etc. have good biocompatibility, film-forming properties, and viscosity, have certain antibacterial and anti-inflammatory effects, and can also promote the regeneration, repair and healing of wound tissues. The polysaccharide is compounded into the mussel mucin composite hydrogel to play a synergistic role, and cross-linking occurs between the polysaccharides, and the two are tightly combined to form a composite hydrogel dressing product with higher adhesion. Among them, the center of dextran is a hydrophilic hydroxyl group. This hydrophilic property makes dextran have better adhesion to cells. At the same time, dextran can further enhance the adhesion between mussel mucin and the complex by improving the wettability of the surface or providing additional adhesion sites.

[0036] Chitosan molecules contain a large number of amino and hydroxyl groups, which enable chitosan to form hydrogen bonds and ionic bonds with dopa groups and lysine in mussel mucin, thereby enhancing adhesion. This synergistic effect makes the adhesion interface stronger and more stable.

[0037] Hyaluronic acid has a negative charge, while mussel mucin has a positive charge. When they are mixed together, a strong electrostatic interaction occurs, which can enhance their adhesion properties. In addition, the negative charge of hyaluronic acid can also form ionic bonds with the positive charge in mussel mucin, further strengthening the adhesion layer. In addition, in the mixed system, the carboxyl group (-COOH) of hyaluronic acid may also bond with the amino group (-NH 2 ) or hydroxyl (-OH) to form intermolecular crosslinks such as covalent bonds or hydrogen bonds. These crosslinked structures can increase the strength and stability of the adhesion layer.

[0038] Chitosan and mussel mucin are complementary in anti-inflammatory effect, they can jointly inhibit the release of inflammatory mediators and reduce leukocyte migration, thereby enhancing the anti-inflammatory effect. Glucan, hyaluronic acid and mussel mucin have synergistic effects in inhibiting inflammatory response, they can jointly inhibit the release of inflammatory mediators and reduce the activity of inflammatory cells, thereby further enhancing the anti-inflammatory effect. The present invention compounds polysaccharides into the hydrogel of recombinant mussel mucin, and plays a synergistic role. Cross-linking occurs between recombinant mussel mucin and polysaccharides, and the two are closely combined, effectively improving the adhesion of the compound hydrogel dressing, promoting wound healing, improving anti-inflammatory efficacy, and forming a good protective barrier environment for the repair and healing of the wound surface.

[0039] In some embodiments, the mass ratio of dextran, chitosan and hyaluronic acid is: (5.0-10.0):(5.0-10.0):(0.1-0.5).

[0040] Under the conditions of the optimal ratio of the polysaccharide composition, the synergistic effect of hyaluronic acid and recombinant mussel mucin can promote cell adhesion and proliferation. The positive charge characteristics of recombinant mussel mucin enable it to effectively attract cells, while hyaluronic acid can provide a microenvironment conducive to cell growth and proliferation. This synergistic effect helps to accelerate the tissue repair and regeneration process. Both dextran and chitosan can provide cells with a microenvironment conducive to growth and proliferation. At the same time, they can also promote cell migration and differentiation, thereby accelerating the tissue repair and regeneration process.

[0041] In some embodiments, the molecular weight of the dextran is 400-600 kDa, the molecular weight of the chitosan is 40-60 kDa, and the molecular weight of the hyaluronic acid is 8-12 kDa.

[0042] In some embodiments, the thickener is one or more of carbomer, sodium polyacrylate, polyvinyl alcohol, methyl cellulose, hydroxyethyl cellulose, xanthan gum and gelatin.

[0043] In some embodiments, the humectant is one or more of glycerin, propylene glycol, butylene glycol, pentylene glycol, sorbitol and polyethylene glycol.

[0044] In some embodiments, the pH adjuster is one of triethanolamine, aminomethyl propanol, sodium hydroxide and potassium hydroxide.

[0045] In some embodiments, the preservative is one or more of sodium benzoate, benzyl alcohol, methylparaben, sodium methylparaben, sodium propylparaben, phenoxyethanol and potassium sorbate.

[0046] In some embodiments, the metal ion chelator is one or more of low molecular weight dextran, ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, and sodium calcium ethylenediaminetetraacetate.

[0047] The embodiment of the present invention also provides a method for preparing the composite hydrogel dressing based on recombinant mussel mucin, comprising the steps of:

[0048] S1, adding the recombinant mussel mucin freeze-dried powder into water to obtain a recombinant mussel mucin solution;

[0049] S2. Add water, thickener and polysaccharide composition into a vacuum homogenizing stirring tank, heat to 60-85° C., and stir at 200-350 rpm for 20-70 minutes to obtain a first mixed solution;

[0050] S3, adding a moisturizer and a metal ion chelating agent to the first solution, stirring and mixing at a rate of 200-400 rpm, and homogenizing at a stirring rate of 2000-5000 rpm for 5-15 minutes to obtain a second mixed solution;

[0051] S4, cooling the second mixed solution to 30-40° C., adding the recombinant mussel mucin solution, and stirring and mixing at a speed of 200-400 rpm to obtain a third mixed solution;

[0052] S5. Add a preservative to the third mixed solution, stir and mix, then add a pH adjuster and water, stir and mix at 200-400 rpm to obtain the composite hydrogel dressing based on recombinant mussel mucin.

[0053] In some embodiments, the pH value of the recombinant mussel mucin-based composite hydrogel dressing is between 5.0 and 6.5.

[0054] The following describes it in detail through specific embodiments.

[0055] Example 1

[0056] The composite hydrogel dressing based on recombinant mussel mucin comprises the following raw material components in parts by weight: 0.05 parts of recombinant mussel mucin, 0.5 parts of carbomer, 10.0 parts of dextran, 10.0 parts of chitosan, 0.1 parts of hyaluronic acid, 2.0 parts of glycerol, 2.0 parts of polyethylene glycol, 0.05 parts of disodium ethylenediaminetetraacetate, 0.5 parts of triethanolamine, 0.1 parts of sodium benzoate, and 74.7 parts of purified water.

[0057] The preparation method of the composite hydrogel dressing based on recombinant mussel mucin comprises the following steps:

[0058] Step S1: adding 0.05 parts of recombinant mussel mucin freeze-dried powder to 10 parts of purified water solution to obtain a recombinant mussel mucin solution.

[0059] Step S2: 70.0 parts of water were taken and added into a vacuum homogenizing stirring tank, 0.5 parts of carbomer, 5.0 parts of dextran, 5.0 parts of chitosan and 0.1 parts of hyaluronic acid were added into the purified water, and the mixture was stirred at 300 rpm and heated to 80° C. to dissolve all the solid powder, and the stirring was continued for 30 minutes, and the heating was turned off;

[0060] Step S3: accurately weighing 2.0 parts of glycerol, 2.0 parts of polyethylene glycol (400), and 0.05 parts of disodium ethylenediaminetetraacetate in the prescribed amount, adding them to the solution obtained in step S2, stirring at a speed of 300 rpm to fully mix the materials, and then homogenizing at a stirring speed of 3000 rpm for 10 minutes to obtain a mixed solution;

[0061] Step S4: cooling the mixed solution obtained in step S3 to 30° C., adding the recombinant mussel mucin solution in step S1, and stirring at a speed of 300 rpm to fully mix the materials to obtain a new mixed solution;

[0062] Step S5: accurately weigh 0.1 parts of sodium benzoate and add it to the mixed solution obtained in step S4, and then stir at a speed of 300 rpm to fully mix the materials to obtain a new mixed solution;

[0063] Step S6: accurately weigh the prescribed amount of triethanolamine and add it to the mixed solution obtained in step S5, add water to the formula to 100 parts by weight, and then stir at a speed of 200-400 rpm to fully mix the materials to obtain a material body with a pH value between 5.5 and 6.5.

[0064] Step S7: After multiple vacuum degassing and electron beam irradiation sterilization, a composite hydrogel dressing based on recombinant mussel mucin can be obtained.

[0065] Example 2

[0066] The composite hydrogel dressing based on recombinant mussel mucin comprises the following raw material components in parts by weight: 0.03 parts of recombinant mussel mucin, 0.5 parts of carbomer, 5.0 parts of dextran, 5.0 parts of chitosan, 0.1 parts of hyaluronic acid, 2.0 parts of glycerol, 1.0 parts of polyethylene glycol, 0.05 parts of disodium ethylenediaminetetraacetate, 0.5 parts of triethanolamine, 0.1 parts of sodium benzoate, and 85.72 parts of purified water.

[0067] The preparation method of the hydrogel dressing based on recombinant mussel mucin in this example is the same as that in Example 1.

[0068] Example 3

[0069] The composite hydrogel dressing based on recombinant mussel mucin comprises the following raw material components in parts by weight: 0.1 parts of recombinant mussel mucin, 0.5 parts of carbomer, 5.0 parts of dextran, 5.0 parts of chitosan, 0.2 parts of hyaluronic acid, 2.0 parts of glycerol, 2.0 parts of polyethylene glycol, 0.05 parts of disodium ethylenediaminetetraacetate, 0.5 parts of triethanolamine, 0.1 parts of sodium benzoate, and 84.55 parts of purified water.

[0070] The preparation method of the hydrogel dressing based on recombinant mussel mucin in this example is the same as that in Example 1.

[0071] Comparative Example 1

[0072] The difference between this comparative example and Example 1 is that no glucan is added.

[0073] Specifically, the hydrogel dressing based on recombinant mussel mucin contains the following raw material components in parts by weight: 0.05 parts of recombinant mussel mucin, 0.5 parts of carbomer, 10.0 parts of chitosan, 0.1 parts of hyaluronic acid, 2.0 parts of glycerol, 2.0 parts of polyethylene glycol, 0.05 parts of disodium ethylenediaminetetraacetic acid, 0.5 parts of triethanolamine, 0.1 parts of sodium benzoate, and 84.7 parts of purified water.

[0074] The preparation method of the hydrogel dressing based on recombinant mussel mucin in this comparative example is the same as that in Example 1.

[0075] Comparative Example 2

[0076] The difference between this comparative example and Example 1 is that chitosan is not added.

[0077] Specifically, the hydrogel dressing based on recombinant mussel mucin contains the following raw material components in parts by weight: 0.05 parts of recombinant mussel mucin, 0.5 parts of carbomer, 10.0 parts of dextran, 0.1 parts of hyaluronic acid, 2.0 parts of glycerol, 2.0 parts of polyethylene glycol, 0.05 parts of disodium ethylenediaminetetraacetic acid, 0.5 parts of triethanolamine, 0.1 parts of sodium benzoate, and 84.7 parts of purified water.

[0078] The preparation method of the hydrogel dressing based on recombinant mussel mucin in this comparative example is the same as that in Example 1.

[0079] Comparative Example 3

[0080] The difference between this comparative example and Example 1 is that hyaluronic acid is not added.

[0081] Specifically, the hydrogel dressing based on recombinant mussel mucin contains the following raw material components in parts by weight: 0.05 parts of recombinant mussel mucin, 0.5 parts of carbomer, 10.0 parts of dextran, 10.0 parts of chitosan, 2.0 parts of glycerol, 2.0 parts of polyethylene glycol, 0.05 parts of disodium ethylenediaminetetraacetic acid, 0.5 parts of triethanolamine, 0.1 parts of sodium benzoate, and 74.8 parts of purified water.

[0082] The preparation method of the hydrogel dressing based on recombinant mussel mucin in this comparative example is the same as that in Example 1.

[0083] Comparative Example 4

[0084] The difference between this comparative example and Example 2 is that other polysaccharides are used to replace dextran and chitosan.

[0085] Specifically, the hydrogel dressing based on recombinant mussel mucin contains the following raw material components in parts by weight: 0.03 parts of recombinant mussel mucin, 0.5 parts of carbomer, 5.0 parts of trehalose, 5.0 parts of corn starch, 0.2 parts of hyaluronic acid, 2.0 parts of glycerol, 1.0 parts of polyethylene glycol, 0.05 parts of disodium ethylenediaminetetraacetic acid, 0.5 parts of triethanolamine, 0.1 parts of sodium benzoate, and 85.72 parts of purified water.

[0086] The preparation method of the hydrogel dressing based on recombinant mussel mucin in this comparative example is the same as that in Example 1.

[0087] Comparative Example 5

[0088] The difference between this comparative example and Example 2 is that other polysaccharides are used to replace dextran and chitosan.

[0089] Specifically, the hydrogel dressing based on recombinant mussel mucin contains the following raw material components in parts by weight: 0.03 parts of recombinant mussel mucin, 0.5 parts of carbomer, 5.0 parts of cellulose, 5.0 parts of loofah polysaccharide, 0.2 parts of hyaluronic acid, 2.0 parts of glycerol, 1.0 parts of polyethylene glycol, 0.05 parts of disodium ethylenediaminetetraacetic acid, 0.5 parts of triethanolamine, 0.1 parts of sodium benzoate, and 85.72 parts of purified water.

[0090] The preparation method of the hydrogel dressing based on recombinant mussel mucin in this comparative example is the same as that in Example 1.

[0091] Comparative Example 6

[0092] The difference between this comparative example and Example 3 is that the ratio of the polysaccharide composition and the recombinant mussel mucin is not within the formula range.

[0093] Specifically, the recombinant mussel mucin hydrogel dressing contains the following raw material components in parts by weight: 0.1 parts of recombinant mussel mucin, 0.5 parts of carbomer, 10.0 parts of dextran, 20.0 parts of chitosan, 0.5 parts of hyaluronic acid, 2.0 parts of glycerol, 2.0 parts of polyethylene glycol, 0.05 parts of disodium ethylenediaminetetraacetic acid, 0.5 parts of triethanolamine, 0.1 parts of sodium benzoate, and 64.25 parts of purified water.

[0094] The preparation method of the hydrogel dressing based on recombinant mussel mucin in this comparative example is the same as that in Example 1.

[0095] Test Example 1

[0096] In order to verify that the formula composition of the present invention can effectively improve the adhesion of the compound hydrogel dressing based on recombinant mussel mucin, the compound hydrogel dressing based on recombinant mussel mucin prepared in the above examples and comparative examples was first subjected to an adhesion test.

[0097] The specific steps are as follows: take two metal plates (2024t3 plates, length * width * thickness: 100mm * 25mm * 1.6mm, punch holes on both sides), apply 12μL of the test liquid (250 square millimeters) on the lower plate, slowly cover the upper plate, and then press the pressing plate (weight 600g) as soon as possible, and dry it in an oven at 45℃. After 48h, take out the sample, remove the pressing plate, let it stand for 0.5h to cool to room temperature, and clamp the sample with a universal tensile machine fixture to ensure that the length direction of the sample is parallel to the tensile direction. Stretch at a speed of 5±1mm / min to make the shear force change between 8.3-9.8MPa. After the sample breaks, the maximum tensile shear strength is calculated using the maximum shear force. Shear force = tension / contact area.

[0098] The experimental results are shown in Table 1:

[0099] Table 1

[0100]

[0101]

[0102] As can be seen from Table 1, compared with Comparative Examples 1-3, the composite hydrogel dressing based on recombinant mussel mucin in Example 1 has higher adhesion. Compared with Comparative Example 1, Example 1 adds more dextran, and the center of dextran is a hydrophilic hydroxyl group. This hydrophilic property makes dextran have better adhesion to cells. At the same time, dextran can further enhance the adhesion between mussel mucin and the composite by improving the wettability of the surface or providing additional adhesion sites.

[0103] Compared with Example 1, Comparative Example 2 added less chitosan. Chitosan molecules contain a large number of amino and hydroxyl groups. These functional groups enable chitosan to form hydrogen bonds and ionic bonds with the dopa group, lysine, etc. in the recombinant mussel mucin, thereby enhancing adhesion. This synergistic effect makes the adhesion interface stronger and more stable.

[0104] Comparative Example 3 adds less hyaluronic acid than Example 1, and hyaluronic acid has a negative charge, while mussel mucin has a positive charge. When they are mixed together, strong electrostatic interaction occurs, and this interaction can enhance their adhesion properties. In addition, the negative charge of hyaluronic acid can also form an ionic bond with the positive charge in mussel mucin, further reinforcing the adhesive layer. In addition, the carboxyl group (-COOH) of hyaluronic acid may also react with the amino group (-NH2) or hydroxyl group (-OH) in the recombinant mussel mucin to form intermolecular crosslinks such as covalent bonds or hydrogen bonds. These cross-linked structures can increase the strength and stability of the adhesive layer.

[0105] The proportions of the polysaccharide compositions in Example 3 and Comparative Example 6 are different, but the difference in adhesion is not significant. This is because the increase in adhesion of the polysaccharide composition to the composite hydrogel dressing based on recombinant mussel mucin is not proportional. For example, when the chitosan concentration is too high, the increase in adhesion will slow down and increase the cost.

[0106] Test Example 2

[0107] In order to verify whether the formula composition of the present invention can effectively improve the cell adhesion of the recombinant mussel mucin hydrogel dressing after the adhesion force is increased, the composite hydrogel dressing based on recombinant mussel mucin prepared in the above Examples 1-2 and Comparative Examples 1-5 was subjected to a cell adhesion test.

[0108] Experimental methods:

[0109] Three 24-well plates were used, each of which was divided into 6 groups. The coverslip coated with poly-lysine was used as the control group, and 100 μL of the hydrogel dressings of Examples 1-2 and Comparative Examples 1-5 were taken from the rest. The cells were 1×103 cells / cm 2 HFF cells were seeded at a density of 1.5 μg / ml and placed at 37°C with 5% CO 2 Cultivation under floating tank;

[0110] After 6h, 12h, and 18h ​​of co-culture, the well plates were taken, the culture medium was discarded, and the well plates were washed once with PBS buffer; 4% paraformaldehyde was added, fixed at room temperature for 20min, and the paraformaldehyde was discarded; the well plates were washed three times with PBS solution, and the PBS solution was discarded; DAPI staining solution was added, and staining was carried out at room temperature and away from light for 8min; the DAPI staining solution was aspirated, the well plates were washed three times with PBS solution, the suspended cells were removed, and the well plates were placed under a fluorescence microscope to count the number of cells stained with DAPI staining solution and attached to the hydrogel dressing.

[0111] The experimental results are shown in Table 2:

[0112] Table 2

[0113]

[0114] As can be seen from Table 2, the cell adhesion of Example 1 is significantly higher than that of Comparative Examples 1-3, which is positively correlated with the adhesion results. The cell adhesion of Example 2 is also higher than that of Comparative Examples 4-5, because polysaccharides such as trehalose and luffa polysaccharide have little effect on the increase of the adhesion of the composite, while polysaccharides such as cellulose and corn starch can enhance the adhesion, but due to their inertness, the enhancement of cell adhesion is not obvious.

[0115] Test Example 3

[0116] The polysaccharide composition with the preferred ratio is compounded with the recombinant mussel mucin, which can have a synergistic anti-inflammatory effect with the mussel mucin. The compounded hydrogel dressing based on recombinant mussel mucin prepared in the above Examples 1-2 and Comparative Examples 1-5 was tested for its anti-inflammatory efficacy.

[0117] Experimental methods:

[0118] Tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) are the most common and important inflammatory mediators in the inflammatory response process, which can cause inflammatory symptoms such as redness, swelling, heat and pain. By reducing the content of these factors, the degree of inflammatory response can be controlled. The experiment used lipopolysaccharide (LPS) to intervene in macrophages to establish an inflammatory cell model, and the anti-inflammatory effect of the hydrogel dressing was evaluated by measuring the content of TNF-α and IL-6 therein. The culture medium of Examples 1-2 and Comparative Examples 1-5 and LPS (1 μg / mL) were added to the test group, and the cell culture medium containing LPS (1 μg / mL) was added to the negative control group. The content of TNF-α and IL-6 was detected by ELISA detection kit. The test results are shown in Table 3.

[0119] The experimental results are shown in Table 3:

[0120] Table 3

[0121]

[0122]

[0123] It can be seen from Table 3 that after the polysaccharide composition with the preferred ratio is compounded with the recombinant mussel mucin, the anti-inflammatory effect of the embodiment is better than that of the compound hydrogel dressing based on recombinant mussel mucin prepared in the comparative example. This is because chitosan and mussel mucin are complementary in anti-inflammatory effect, and they can jointly inhibit the release of inflammatory mediators and reduce the migration of leukocytes, thereby enhancing the anti-inflammatory effect. Glucan, hyaluronic acid and mussel mucin have a synergistic effect in inhibiting inflammatory response, and they can jointly inhibit the release of inflammatory mediators and reduce the activity of inflammatory cells, thereby further enhancing the anti-inflammatory effect.

[0124] Test Example 4

[0125] In order to verify that the polysaccharide composition with the preferred ratio and the recombinant mussel mucin are compounded to facilitate wound healing, the compounded hydrogel dressings based on recombinant mussel mucin prepared in the above Example 1 and Comparative Examples 1-3 were subjected to skin injury healing tests.

[0126] Experimental method: 4 hours before the experiment, the hair of healthy SD rats was removed on both sides of the spine of the animal's back, which was used as the experimental and observation site. The surgical area was disinfected with iodine and 75% ethanol. Figure 1As shown, a skin wound of 2 cm*2 cm in size and deep to the superficial dermis was made in the center of the animal's back by surgery. The hydrogel dressing prepared in the above Example 1 and Comparative Examples 1-3 was applied until the wound was completely covered, and then covered with a PU film and bandaged, twice a day, with an interval of 6 hours, from the 0th day after surgery to the 14th day after surgery. The wound area was measured and recorded after surgery, and the wound area was measured and recorded after administration on the 7th and 14th days after surgery, and the wound healing rates on the 7th and 14th days were calculated.

[0127] The experimental results are shown in Table 4:

[0128] Table 4

[0129]

[0130] It can be seen from Table 4 that under the preferred ratio of polysaccharide composition and specific mussel mucin type, the healing effect of the embodiment is better than that of the composite hydrogel dressing based on recombinant mussel mucin prepared in the comparative example. This is because the synergistic effect of hyaluronic acid and mussel mucin can also promote cell adhesion and proliferation. The positive charge characteristics of mussel mucin enable it to effectively attract cells, while hyaluronic acid can provide a microenvironment that is conducive to cell growth and proliferation. This synergistic effect helps to accelerate the tissue repair and regeneration process. Both dextran and chitosan can provide cells with a microenvironment that is conducive to growth and proliferation. At the same time, they can also promote cell migration and differentiation, thereby accelerating the tissue repair and regeneration process.

[0131] like Figure 2 As shown, in the healing process of the composite hydrogel dressing based on recombinant mussel mucin in Example 1, the healing rate of Example 1 reached 84.3% on the 7th day after surgery and reached 99.0% on the 14th day, which was basically completely healed. Figure 3-5 As shown, during the healing process of the composite hydrogel dressings based on recombinant mussel mucin of Comparative Examples 1-3, the healing rates were lower than those of Example 1 at the 7th and 14th days after surgery.

[0132] Test Example 5

[0133] The dressings of Examples 1-3 were subjected to pyrogen, cytotoxicity, irritation, and acute toxicity-related biocompatibility tests according to the GB / T 16886 series of standards. The test results are shown in Table 5.

[0134] Table 5

[0135]

[0136]

[0137] All the examples satisfy the above biocompatibility test, indicating that the composite hydrogel dressing based on recombinant mussel mucin prepared in the examples of the present invention has good biocompatibility.

[0138] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A composite hydrogel dressing based on recombinant mussel mucin, characterized in that: The raw material components include, by weight: 0.01-0.3 parts of recombinant mussel mucin, 0.1-2.0 parts of thickener, 10-21 parts of polysaccharide composition, 1-8 parts of moisturizer, 0.01-0.5 parts of pH regulator, 0.05-0.2 parts of preservative, 0.05-0.2 parts of metal ion chelator and 68-88 parts of purified water; The polysaccharide composition comprises dextran, chitosan and hyaluronic acid.

2. The composite hydrogel dressing based on recombinant mussel mucin according to claim 1, characterized in that: The mass ratio of the dextran, chitosan and hyaluronic acid is: (5.0-10.0):(5.0-10.0):(0.1-0.5).

3. The composite hydrogel dressing based on recombinant mussel mucin according to claim 2, characterized in that: The molecular weight of the dextran is 400-600 kDa, the molecular weight of the chitosan is 40-60 kDa, and the molecular weight of the hyaluronic acid is 8-12 kDa.

4. The composite hydrogel dressing based on recombinant mussel mucin according to claim 1, characterized in that: The thickener is one or more of carbomer, sodium polyacrylate, polyvinyl alcohol, methyl cellulose, hydroxyethyl cellulose, xanthan gum and gelatin.

5. The composite hydrogel dressing based on recombinant mussel mucin according to claim 1, characterized in that: The moisturizing agent is one or more of glycerin, propylene glycol, butylene glycol, pentylene glycol, sorbitol and polyethylene glycol.

6. The composite hydrogel dressing based on recombinant mussel mucin according to claim 1, characterized in that: The pH regulator is one of triethanolamine, aminomethyl propanol, sodium hydroxide and potassium hydroxide.

7. The composite hydrogel dressing based on recombinant mussel mucin according to claim 1, characterized in that: The preservative is one or more of sodium benzoate, benzyl alcohol, methylparaben, sodium methylparaben, sodium propylparaben, phenoxyethanol and potassium sorbate.

8. The composite hydrogel dressing based on recombinant mussel mucin according to claim 1, characterized in that: The metal ion chelating agent is one or more of low molecular weight dextran, ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate and sodium calcium ethylenediaminetetraacetate.

9. A method for preparing a composite hydrogel dressing based on recombinant mussel mucin according to any one of claims 1 to 8, characterized in that: Includes steps: S1, adding the recombinant mussel mucin freeze-dried powder into water to obtain a recombinant mussel mucin solution; S2. Add water, thickener and polysaccharide composition into a vacuum homogenizing stirring tank, heat to 60-85° C., and stir at 200-350 rpm for 20-70 minutes to obtain a first mixed solution; S3, adding a moisturizer and a metal ion chelating agent to the first solution, stirring and mixing at a rate of 200-400 rpm, and homogenizing at a stirring rate of 2000-5000 rpm for 5-15 minutes to obtain a second mixed solution; S4, cooling the second mixed solution to 30-40° C., adding the recombinant mussel mucin solution, and stirring and mixing at a speed of 200-400 rpm to obtain a third mixed solution; S5. Add a preservative to the third mixed solution, stir and mix, then add a pH adjuster and water, stir and mix at 200-400 rpm to obtain the composite hydrogel dressing based on recombinant mussel mucin.

10. The method for preparing the composite hydrogel dressing based on recombinant mussel mucin according to claim 9, characterized in that: The pH value of the composite hydrogel dressing based on recombinant mussel mucin is between 5.0 and 6.5.

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